The invention generally relates to label-free imaging systems, and relates in particular to label-free microscopy and micro-spectroscopy imaging systems employing efficient detection of signals of interest in non-linear optical microscopy and micro-spectroscopy imaging systems and methods.
Conventional label-free optical imaging techniques include, for example, infrared microscopy, Raman microscopy, coherent anti-Stokes Raman scattering (CARS) microscopy and modulation transfer microscopy. Micro-spectroscopy generally involves capturing a spectrum from a microscopic volume in a sample, while microscopy generally involves capturing an image as well as scanning such that multiple images are captured to form picture elements (pixels) of a microscopy image.
Infrared microscopy involves directly measuring the absorption of vibrational excited states in a sample, but such infrared microscopy is generally limited by poor spatial resolution due to the long wavelength of infrared light, as well as by a low penetration depth due to a strong infrared light absorption by the water in biological samples.
Raman microscopy records the spontaneous inelastic Raman scattering upon a single (ultraviolet, visible or near infrared) continuous wave (CW) laser excitation. Raman microscopy has improved optical resolution and penetration depth as compared to infrared microscopy, but the sensitivity of Raman microscopy is rather poor because of the very low spontaneous Raman scattering efficiency (a Raman scattering cross section is typically on the order of 10−30 cm2). Although spontaneous Raman emissions is emitted in all directions, the low spontaneous Raman scattering efficiency results in long averaging times per image, which limits the biomedical application of Raman microscopy.
FIG. 1A, for example, shows the generation of incoherent emission 10 in a microscopy imaging system that is emitted in all directions from a focal volume 12 after one or more excitation fields are directed toward the focal volume 12 through an objective lens 14. Such a microscopy imaging system may employ one or two-photon excited fluorescence as well as spontaneous Raman emission. As may be seen in FIG. 1A, the emission is produced in the forward as well as the reverse (epi) direction.
CARS microscopy, which uses two pulsed laser excitation beams (pump and Stokes beams), significantly increases the absolute scattering signal due to the coherent excitation. The CARS process, however, also excites a high level of background from the vibrationally non-resonant specimen. Such a non-resonant background not only distorts the CARS spectrum of the resonant signal from dilute sample but also carries the laser noise, significantly limiting the application of CARS microscopy on both spectroscopy and sensitivity perspectives.
One approach to reduce the non-resonant background field in CARS microscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. For example, U.S. Pat. No. 6,798,507 discloses a system in which the pump and Stokes beams are properly polarized and a polarization sensitive detector is employed. Another approach to reducing the non-resonant background field involves detecting the anti-Stokes field in a reverse direction. U.S. Pat. No. 6,809,814 discloses a system in which a CARS signal is received in the reverse direction (epi-direction) from the sample. For transparent samples, however the epi directed signal is significantly smaller than the forward directed signal.
FIG. 1B shows the generation of a signal of interest of a coherent emission microscopy system, such as CARS, second harmonic generation SHG or third harmonic generation THG. The coherent emission 20 in such a microscopy imaging system is emitted primarily in the forward direction as shown at 22, while a much lesser intensity is directed in the reverse (epi) direction as shown at 24 from a focal volume 26 after one or more excitation fields are directed toward the focal volume 26 through an objective lens 28.
Modulation transfer microscopy and micro-spectroscopy imaging systems such as stimulated Raman scattering, stimulated emission, one photon and two photon photo-thermal imaging, two-color two-color absorption, stimulated Brillouin scattering and cross-phase modulation imaging generally involve reliance on the non-linear interaction of two laser beams within a sample, and detection of a characteristic, such as gain or loss, of one of the excitation beams. This is in contrast to detecting a newly generated (new frequency) emission signal as is done, for example, in one-photon and two-photon excited fluorescence, spontaneous Raman scattering, coherent anti-Stokes Raman scattering (CARS), second harmonic generation, (SHG) and third harmonic generation (THG).
Such modulation transfer microscopy and micro-spectroscopy techniques require a detection scheme that provides for detection of a relatively small signal (e.g., a small gain and loss signal) on top of noisy lasers. This is generally achieved based on modulation transfer, that is by modulating a feature of one of the laser excitation beams and measuring the signal of interest with high sensitivity. In particular, the modulation transfers to the other excitation beam due to non-linear interaction within the sample, which facilitates detection of the signal of interest using a modulation sensitive detector. If the modulation frequency is chosen to be faster than the laser noise (e.g., greater than about 200 kHz), shot-noise limited sensitivity may be achieved. Such modulation schemes are readily compatible with beam-scanning microscopy and micro-endoscopy, video-rate imaging speeds, and multiplex excitation schemes.
FIG. 1C shows the generation of a signal of interest of a modulation transfer microscopy or spectroscopy imaging system, such as stimulated Raman scattering, stimulated emission, one photon and two photon photo-thermal imaging, two-color two-color absorption, stimulated Brillouin scattering and cross-phase modulation imaging. One or more excitation fields are directed toward the focal volume 34 through an objective lens 36. As in modulation transfer microscopy, gain or loss of the excitation beams is detected, the emission is effectively only directed into the forward direction. The modulation transfer emission 30 in such a microscopy imaging system is emitted from a focal volume 34 therefore only in the forward direction as shown at 32.
While modulation transfer microscopy and spectroscopy provides high sensitivity in a forward direction, certain applications would benefit from the ability to detect a modulation transfer signal (shown at 30 in FIG. 1C) in the epi direction. Incoherent emission (shown at 10 in FIG. 1A) and to a lesser extent, coherent emission (shown at 20 in FIG. 1B) provide epi-directed emission signals from the focal volume, but have other limitations as discussed above.
There remains a need, therefore, for a microscopy and micro-spectroscopy imaging systems that provide high sensitivity in the epi direction. There is further a need to provide images at a sufficiently high rate that video imaging may be provided. CARS microscopy in certain applications, however, suffers from spectral distortion, limited sensitivity due to unwanted non-resonant background as discussed above, non-linear concentration dependence and coherent image artifacts, thereby limiting quantitative interpretation at video-rate speeds.